IJCRT2205468

www.ijcrt.
org © 2022 IJCRT | Volume 10, Issue 5 May 2022 | ISSN: 2320-2882
DEVELOPMENT AND VALIDATION OF

MOLNUPIRAVIR IN PHARMACEUTICAL
DOSAGE FORM
ALAGIYA KAJAL JAYANTIBHAI
Guided By
Mrs. FORAM PATEL
M. Pharm.
Assistant Professor
Abstract:
The present study was focused to develop a simple, precise, accurate and cost effective RP-HPLC method for
estimation of Molnupiravir in Pharmaceutical dosage form. The chromatographic method was carried out using
Kromasil 100-5-C18 (150 mm x 4.6 mm) 5µ column with mobile phase Buffer solution (0.1%
Orthophosphoric acid): Acetonitrile in ratio of 85:15 % v/v. The flow rate was set 1.0 ml/min with 10 µL injection
volume. Total run time 10 min. Detection was carried out at the wavelength of 235 nm. The detector response was
linear in the concentration range of 50 – 150µg/ml with a correlation coefficient of 0.999. . The developed analytical
method was validated according to the ICH guideline. The developed method is successfully applied for estimation
of Molnupiravir in Pharmaceutical dosage form
Keywords: Molnupiravir, RP-HPLC, Method Development, Validation.
1. Introduction to Corona Virus1-5
1.1 SARS-CoV-2/COVID-19
 2019 Novel Coronavirus (SARS-CoV-2), causing coronavirus disease 2019 (COVID-19), is a virus
identified as the cause of an outbreak of respiratory illness first detected in Wuhan, China.
 Initially, the patients were believed to have contracted the virus from seafood/animal markets which
suggested animal-to-human spread.
 The growing number of patients however, suggest that human-to-human transmission is actively
occurring.
IJCRT2205468 International Journal of Creative Research Thoughts (IJCRT) www.ijcrt.org e186

www.ijcrt.org © 2022 IJCRT | Volume 10, Issue 5 May 2022 | ISSN: 2320-2882
Signs and Symptoms:

• Fever, Tiredness, Cough, Loss of test or smell, Difficulty of breathing, Muscle ache, Chills, Sore
throat, Runny nose, Headache, Chest pain, Pink eye (Conjuctivities)
Causes:
 The virus that causes COVID-19 spreads easily among people, and more continues to be discovered
over time about how it spreads. Data has shown that it spreads mainly from person to person among
those in close contact (within about 6 feet, or 2 meters). The virus spreads by respiratory droplets
released when someone with the virus coughs, sneezes, breathes, sings or talks. These droplets can
be inhaled or landin the mouth, nose or eyes of a person nearby.
 In some situations, the COVID-19 virus can spread by a person being exposed to small droplets or
aerosols that stay in the air for several minutes or hours called airborne transmission. It's not yet
known how common it is for the virus to spread this way.
 It can also spread if a person touches a surface or object with the virus on it and thentouches his or
her mouth, nose or eyes, although this isn't considered to be a main way it spreads. Some
reinfections of the virus that causes COVID-19 have happened, but these have been uncommon
1.2 CORONA VIRUS:
 Coronaviruses are a family of viruses that cause illness such as respiratory diseases or
gastrointestinal diseases. Respiratory diseases can range from the common cold to more severe
diseases
Ex:
Middle East Respiratory Syndrome (MERS-CoV)

Severe Acute Respiratory Syndrome (SARS-CoV)
• Coronavirus (nCoV) is a new strain that has not been identified in humans previously. Once
scientists determine exactly what coronavirus it is, they give ita name (as in the case of COVID-
19, the virus causing it is SARS-CoV-2).
 Coronaviruses got their name from the way that they look under a microscope. The virus consists
of a core of genetic material surrounded by an envelope with protein spikes. This gives it the
appearance of a crown. The word Corona means “crown” in Latin.
Coronaviruses are zoonotic, meaning that the viruses are transmitted between animals and
humans. It has been determined that MERS-CoV was transmittedfrom dromedary camels to
humans and SARS-CoV from civet cats to humans. The source of the SARS-CoV-2 (COVID-
19) is yet to be determined, butinvestigations are ongoing to identify the zoonotic source to.

1.3 DRUGS USED IN CORONAVIRUS

 Hydroxychloroquine
 Favipiravir
 Remdesivir
 Molnupiravir
 Tocilizumab
 Itolizumab (only preliminary results available)
 Steroids: Dexamethasone, Methylprednisolone
 Low molecular weight Heparin

 Antibiotics - Azithromycin, Ivermectin
 Convalescent Plasma Therapy
2. DRUG PROFILE 6
INTRODUCTION
Name Molnupiravir
Official in Not Official in any Pharmacopoeia
Molnupiravir is an orally bioavailable

isopropylester cytidine analog being
investigated to treat COVID-19. Molnupiravir is
hydrolyzed in vivo to N4-hydroxycytidine,
which is phosphorylated in tissue to the active
5’-triphosphate form, and incorporated into the
genome of new virions, resulting in the
Description & Mechanism of accumulation of inactivating mutations, known
action as viral error catastrophe. A remdesivir resistant
mutant mouse hepatitis virus has also been
shown to have increased sensitivity to N4-
hydroxycytidine. N4-Hydroxycytidine
Molnupiravir is hydrolyzed to N4-
hydroxycytidine, which distributes into tissues.2
Once inside cells, N4-hydroxycytidine is
phosphorylated to the 5'-triphosphate form.

Structure
Chemical Formula C13H19N3O7

Mol. Weight 329.31 g/mol
[(2R,3S,4R,5R)-3,4-dihydroxy-5-[(4Z)-4-(hydrox
IUPAC Name yimino)-2-oxo-1,2,3,4-tetrahydropyrimidin-1-yl]o
xolan-2-yl]methyl 2-methylpropanoate
Categories Antiviral drug
Solubility Soluble in water, Freely Soluble in methanol,

Ethanol & DMSO.
PROPERTIES
State White crystalline Solid
CAS NO. 2349386-89-4
Melting point 151-153°C
Property Value
Experimental properties Water solubility 5.77 mg/mL
Log P 1.5
pKa 8.21
2.1 INTRODUCTION TO DOSAGE FORM:
Sr. Brand Name Manufacturer Dosage Dose

No. form
1 LAGEVRIO Merck Sharp & Capsule Molnupiravir 200 mg

200mg , Dohme Corp.
400mg Molnupiravir 400 mg

3 Experimental Work
3.1 METHOD DEVELOPMENT FOR ESTIMATION OF MOLNUPIRAVIR BY

RP-HPLC.
3.1.1 Selection of wavelength
Standard solution of Molnupiravir (10 μg/mL) in Methanol was scanned between 200-400 nm using
UV-visible spectrophotometer. Wavelength was selected from the spectra of above
solution.Molnupiravir show response at 235 nm and 271 nm, but it shows good response at 235 nm
hence 235 nm was selected for further work.
235 nm
271 nm
Figure 3.1: UV Spectrum of Molnupiravir (10µg/ml) showing selection of wavelength detection.

3.1.2 Preparation of standard solutions:
A. Molnupiravir standard stock solution: (1000 μg/ml)
Accurately weighed quantity of 50 mg of Molnupiravir standard was transferred to a 50 mL
volumetric flask. Added about 25 ml of diluent and sonicated to dissolve. Final volume was made up
to the mark with diluent and mixed.
B. Preparation of standard solution of Molnupiravir (100 μg/mL):

Taken 5 mL from the Molnupiravir stock solution and transferred to 50 mL volumetric flask and
volume made up to the mark with diluent and mixed. This solution was used in particular trials.
C. Preparation of standard solution of Molnupiravir (10 μg/mL):

Taken 5 mL from the Molnupiravir stock solution and transferred to 50 mL volumetric flask and
volume made up to the mark with diluent and mixed. This solution was used in selection of
wavelength.

3.1.3 Preparation of synthetic Mixture and placebo mixture
A. Preparation of synthetic mixture

Mixed Molnupiravir, MCC, Polyvinylpyrrolidone K-30, Crossprovidone, Sodium stearyl fumarate,
in proportions of 200:50:10:10:5 respectively in laboratory.
B. Preparation of Placebo
Mixed MCC, Polyvinylpyrrolidone K-30, Crossprovidone, Sodium stearyl fumarate, in proportions
of 50:10:10:5 respectively in laboratory.
3.1.4 Selection of Mobile Phase:

Trail contains various mobile phase which are considered of Methanol, Water, Acetonitrile and Buffer
solution in different proportions and different volumes at different flow rate were tried.
On the basis of various trails the ratio of Buffer solution (0.1% Orthophosphoric acid) and Acetonitrile
(85:15 %V/V) at 1.0 mL/min flow rate, proved to be better than the other mixture in terms of peak
shape, theoretical plate and asymmetry.
A. Preparation of Buffer solution(0.1% Orthophosphoric acid):

Diluted 1.0 ml of Orthophosphoric acid into 1000 ml of Milli-Q water and Mixed.
B. Preparation of Mobile Phase
Prepared a degased Mixture of buffer solution (0.1% Orthophosphoric acid) and acetonitrile in ratio
of 85:15 %V/V and sonicated for 10 min.
C. Preparation of Diluent
Prepared a degased Mixture of Water and Methanol in ratio of 50:50 %V/V and sonicated for 10 min.
D. Preparation of standard solutions (100 μg/mL)

volumetric flask. Added about 25 ml of diluent and sonicated to dissolve. Volume was made up to
the mark with diluent and mixed. Further diluted 5.0 ml of this solution to 50 ml with diluent and
mixed.
E. Preparation of sample solutions (100 μg/mL)

Calculate average net content using 20 capsules. Accurately weighed amount of sample powder
equivalent to 200 mg of Molnupiravir was transferred in to 200 ml volumetric flask. Added about
140 ml of diluent was added and solution was sonicated for 30 min to ensure complete solubilisation
of drug. Then volume was made up to mark with diluent and mixed. Further diluted 5.0 ml of this
solution to 50 ml with diluent and mixed. Filtered solution through 0.45 μm membrane filter paper.

Chromatographic conditions:
Column: Kromasil 100-5-C18 (150 mm x 4.6 mm) 5µ
Mobile Phase: Buffer solution (0.1% Orthophosphoric acid): Acetonitrile
(85:15 % v/v)
Flow Rate: 1.0 ml/min
Detection Wavelength: 235 nm
Injection volume: 10.0 μl
Run time: 10 min
3.1 Chromatogram of Molnupiravir in buffer (0.1% OPA): Acetonitrile (85:15%v/v) (Flow rate-
1.0 ml/min).
 Observed values for system suitability test:

1. Column efficiency (N): Number of theoretical plates observed for Molnupiravir was 8803
2. Symmetry factor (S): Tailing factor observed for Molnupiravir was 1.18
Table 3.1 Results for system suitability test.

Parameters Data observed
Theoretical plates per column 8803
Symmetry factor/Tailing factor 1.18

System suitability / System precision

 Procedure
System suitability and precision were demonstrated by injecting five replicate injections of standard
solution prepared as per the test method. The peak area of analyteof replicate standard injection was
recorded. The theoretical plate and tailing factor for analyte peak were evaluated from standard
solution. The precision was evaluated by computing the relative standard deviation for the peak area
of these replicate injections.
 Acceptance criteria
The column efficiency for analyte peak should not be less than 2000 theoretical plates.
The tailing factor should not be more than 2.0
The relative standard deviation for five replicates standard injections should not be more than 2.0%.
3.1.2.1 Filter compatibility and saturation

 Procedure
The filter saturation was verified by preparing the assay samples with optimized samples preparation
and analyzed the samples by discarding different volume of sample solution. The assay of these
samples was determined.
The difference between the unfiltered and filtered samples should NMT 2.0 %
Table 3.2 Filter Compatibility and Saturation
Discarded volume % of Molnupiravir % Difference

Unfiltered 100.3 -NA-
After 3 ml 99.5 -0.8
After 5 ml 100.2 -0.1
After 7 ml 100.1 -0.2
 Conclusion:
After performing filter saturation study using 0.45 µm Membrane filter which indicates that this
filter is compatible for analysis of sample. We recommended using 0.45 µ membrane filters with
discard volume 5 ml for analysis.
3.1.2.2 Linearity & Range

 Procedure
The linearity of detector response for Molnupiravir was demonstrated by preparing solutions of
Molnupiravir working standard over the range of 50-150 % of standard concentrations. These
solutions were injected into the HPLC system and the area of analyte peak was recorded. A graph of
concentration vs. analyte peak response was plotted. The concentration co efficient between
concentration & analyte peak responseand Y- intercept of the correlation plot was evaluated.

Preparation of Linearity stock solution (1000 ppm)

the mark with diluent and mixed.
50% solution preparation (50 µg/mL): 2.5 mL from stock solution was taken and transferred into
50 mL flask and made up the volume with diluent.
80% solution preparation (80 µg/mL): 4.0 mL from stock solution was taken and transferred into
50 mL flask and made up the volume with diluent.
100% solution preparation (100 µg/mL): 5.0 mL from stock solution was taken and transferred
into 50 mL flask and made up the volume with diluent.
The correlation coefficient should not be less than 0.990.
Y- Intercept was ±2 % of 100 % linearity level response

Table 3.3 Linearity parameter
Linearity Level Conc. of Molnupiravir Peak area of
(%) (µg/ml) Molnupiravir
50 50 1112
80 80 1715
100 100 2218
120 120 2625
150 150 3320
Figure 3.2: Linearity curve of Molnupiravir

3.1.2.3 Method precision

 Procedure
Method precision was demonstrated by preparing six samples as per the test method representing a
single batch. The assay of these samples was determined and the precision and the precision of
method was evaluated by computing the percentage relative standard deviation of assay results.
% RSD for assay of six replicate preparations should not more than 2.0.
Table 3.4 Method precision result
Sample Set % Assay (Molnupiravir)

1 99.6
2 100.6
3 100.0
4 99.0
5 99.5
6 99.2
Average 99.7
% RSD 0.58
 Conclusion:
The low % RSD observed on the assay values indicates that method is precise.
3.1.2.4 Accuracy
 Procedure
The accuracy of the test method was demonstrated by preparing recovery samples i.e. spiking of with
known quantities of API in placebo at the level of 50%, 100%, and 150% of target concentration.
The recovery samples were prepared in triplicate. The above samples were injected and the
percentage recovery for amount added was estimated. The precision of the recovery was determined
by computing the relative standard deviation of triplicate recovery results.
Preparation of recovery solutions:

50% Recovery: Weighed accurately 50 mg API and 42.5 mg of placebo in 100 ml of volumetric
flask.
flask.
flask.
Note: Further procedure for 50%, 100%, and 150% followed as per Sample preparation.
The recovery should be 98.0- 102.0 % and the RSD should NMT 2.0 %

3.1.2.5Accuracy
Table 3.5 Result of Accuracy
Weight Of API API
Sample % % %
Level Placebo Added Recovered
Set Recovery Mean RSD
(mg) (mg) (mg)
Set 01 42.5 50 49.7 99.4
50% Set 02 42.5 50 49.8 99.6 99.8 0.53
Set 03 42.5 50 50.2 100.4
Set 01 42.5 100 101.2 101.2
100% Set 02 42.5 100 99.8 99.8 100.4 0.72
Set 03 42.5 100 100.2 100.2
Set 01 42.5 150 148.1 98.7
150% Set 02 42.5 150 146.3 98.2 98.9 0.82
Set 03 42.5 150 149.9 99.8
 Conclusion:
The result of this study was found to be within the acceptance criteria of method validation (i.e. the
recovery is 98.0 – 102.0 % and the RSD is NMT 2.0 %), this provesthat the test method is accurate
for the estimation of Molnupiravir in Molnupiravir Capsule.
3.1.2.6Specificity
Figure 3.3: Chromatogram of Diluent
Figure 3.4: Chromatogram of Placebo

Figure 3.5: Chromatogram of Molnupiravir Standard
Figure 3.6: Chromatogram of Molnupiravir sample
Figure 3.7: Overlay Chromatogram of Molnupiravir Placebo and Sample

 Conclusion:
The result of the study indicate that there was no interference from blank and placebo with analyte
and method is specific for the estimation of assay of Molnupiravir in Molnupiravir Capsule.
3.1.2.7 Robustness
According to robustness there is minor but deliberate change made in chromatographic Parameters.
 Procedure
Preparation of standard solution (100 ppm)
the mark with diluent and mixed. Further diluted 5.0 ml of this solution to 50 ml with diluent and
mixed.
Change in organic phase ratio of mobile phase: Buffer: ACN (83:17) %v/v and Buffer: ACN
(87:13) %v/v
Change in flow rate: 0.9 ml/min and 1.1 ml/min
3.1.2.8 Robustness
Table 3.6 Robustness parameter
Retention
Theoretical Tailing %
Condition Time
plate factor RSD
(min)
Normal 8103 1.17 5.57 0.27
Organic mobile phase ratio:
7992 1.19 6.81 0.23
[Buffer : ACN (87:13) %v/v]
Organic mobile phase ratio:
8315 1.16 4.72 0.18
[Buffer : ACN (83:17) %v/v]
Flow rate: 0.9 ml/min 8079 1.17 6.20 0.32
Flow rate: 1.1 ml/min 8243 1.15 5.01 0.19

 Conclusion:
Theoretical Plates and Asymmetry value are from the first injection of the system suitability set were
found within the acceptance criteria as per system suitability. So, the study proves the reliability of
test method for minor changes in chromatographic condition. Hence method can be termed as robust.
3.1.2.9 Ruggedness (Intermediate Precision)
To measure of reproducibility test results under the variation in conditions normally expected
different laboratory, different analyst, different instrument and different day.
 Procedure
Ruggedness of method was verified by preparing the standard solution and six replicate of assay
samples as per test procedure on different day and analyzed on HPLC.
% RSD for assay of six replicate preparations should not more than 2.0.
The difference between method precision and Intermediate Precision samples should NMT 3.0 %
3.1.2.10Ruggedness (Intermediate Precision)

Table 3.7 Ruggedness (Intermediate Precision) result
% Assay
Sample Set
(Molnupiravir)
1 98.3
2 99.2
3 99.3
4 100.1
5 97.9
6 98.6
Average 98.9
% RSD 0.80
 Conclusion:
Theoretical Plates and Asymmetry value were found within the acceptance criteria. The low %
RSD observed on the assay values indicates that method is precise. Difference between method
precision and Ruggedness (Intermediate Precision) was found 0.8%. Hence the study proves the
method is rugged for analysis of Molnupiravir in Molnupiravir capsule.

3.2.1.11 Stability of analytical solution

 Procedure
Stability of standard solution and sample solution were established at room temperature(about 250C)
for minimum 24 hours. Standard solution and sample solution were prepared as per test method.
 Acceptance criteria:
The response of standard and sample solution should not differ by more than 2.0% from initial
response for the accepted storage time.
Table 3.8 Stability of analytical solution for standard solution
% deviation from
Time (hrs.) Peak area
initial area
Initial 2199 -
7 HR 2193 0.3
23 HR 2180 0.9
29 HR 2173 1.2
Table 3.9 Stability of analytical solution for sample solution
% deviation from
Time (hrs.) Peak area
initial area
Initial 2190 -
7 HR 2195 -0.2
23 HR 2168 1.0
29 HR 2157 1.5
 Conclusion:
The Standard and Sample solution of Molnupiravir is stable at room temperature (i.e.about
250C) up to 29 hours

3.10 Method Validation Summary
Parameter Molnupiravir
Concentration range (µg/ml) 50 - 150 ppm (50-150%)
Regression equation y = mx + c y = 22.172x – 19.241
Correlation coefficient 0.999

% RSD : 0.11 %
System Suitability Theoretical plates : 8003
Tailing factor : 1.16
Method Precision 99.7 % (% RSD: 0.58%)
50% 99.8 % (% RSD: 0.53 %)
Accuracy 100% 100.4 % (% RSD: 0.72 %)

(%Recovery)
150% 98.9 % (% RSD: 0.82 %)
No interference from blank and placebo

Specificity with analyte so method is specific.
Theoretical Plates and Asymmetry value

Robustness are found within the acceptance criteria.
% RSD found to be less than 2.0 %.
Ruggedness 98.9 % (RSD: 0.80%)

(Intermediate Precision) Difference between method precision and
Intermediate Precision : 0.8%
The Standard and Sample solution is

Stability of analytical solution stable at room temperature (i.e. about
250C) up to 29 hours.

ASSAY OF MOLNUPIRAVIR IN PHARMACEUTICAL DOSAGE FORM BY DEVELOPED RP-

HPLC METHOD:
Table 3.11 Assay of Molnupiravir by developed RP-HPLC method
Label Claim Amount found

Brand Name % Assay
(mg) (mg)
MOLFLU 200.4 100.2
200
(Dr Reddy’S 201.6 100.8
Laboratories LTD) Mean 201.0 100.5
MOLUSAFE 197.6 98.8
200
(Astraea Life 199.4 99.7
sciences PVT LTD Mean 198.5 99.3
SUMMARY & CONCLUSION

8.1 Summary
There is no analytical work has been available regarding HPLC method for Molnupiravir in a
literature. A novel attempt in a field of research has been made to develop and validate assay method
and to demonstrate degradation profile via RP-HPLC.
A simple and rapid RP-HPLC method has been developed for determination of Molnupiravir in
pharmaceutical dosage form. Separation has been achieved using Kromasil 100-5-C18 (150 mm x
4.6 mm) 5µ column using Buffer solution (0.1% OPA): Acetonitrile (85:15, v/v). Detection
wavelength is 235 nm and retention time of Molnupiravir is about 5.50 min with 1.0 ml/min flow
rate..
The RP HPLC method for estimation of Molnupiravir was validated as per ICH Guideline Q2 (R1).
Method Validation Parameters like System Suitability, System Precision, Filter Compatibility,
Method Precision, Linearity, Accuracy, Specificity, Robustness, Ruggedness, and Stability of
analytical solutions were performed. All parameter results are found within the acceptance limit.
8.2 Conclusion
RP-HPLC method has been developed and validated for the estimation of Molnupiravir from
Molnupiravir Capsule dosage form. The method was found to be specific. The present method
has been found to be adequately robust and cost effective. The method was validated as per ICH
guidelines.All parameter and results are found within the acceptance limit.
So, we can conclude that Developed Stability indicating RP-HPLC method is found tobe linear,
specific, accurate, robust, rapid and cost effective. Thus, the proposed method can be used in
routine quality control analysis for estimation of Molnupiravir from Molnupiravir Capsule dosage
form.
4. REFERENCES
1) “Introduction of Corona virus” , Last accessed on September 2020
https:// www.physio-pedia.com/Coronavirus_Diseases_(COVID-19)
2) “Symptoms and causes of Corona virus” , Last accessed on September 2020
https://www.mayoclinic.org/diseases-conditions/coronavirus/symptoms-causes/syc-20479963
3) “Risk factors of Corona virus” , Last accessed on September 2020
https://www.hopkinsmedicine.org/health/conditions-and-diseases/coronavirus/coronavirus-
and-covid19-who-is-at-higher-risk
4) “Pathophysiology of corona virus” , Last accessed on September 2020
https:// www.wikidoc.org?/index.php/coronavirus_pathophysiology
5) “ Drugs used in Corona virus” Last accessed on September 2020
https://www.cnbctv18.com/healthcare/covid-19-treatment-update-here-is-a-list-of-all-drugs-used-
in-india-their-pross-and-cons-6322301.htm
6) “Drug profile for Molnupiravir”, https://go.drugbank.com/drugs/DB15661

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DEVELOPMENT AND VALIDATION OF

ALAGIYA KAJAL JAYANTIBHAI

Keywords: Molnupiravir, RP-HPLC, Method Development, Validation.

1. Introduction to Corona Virus1-5

IJCRT2205468 International Journal of Creative Research Thoughts (IJCRT) www.ijcrt.org e186

Signs and Symptoms:

Middle East Respiratory Syndrome (MERS-CoV)

IJCRT2205468 International Journal of Creative Research Thoughts (IJCRT) www.ijcrt.org e187

1.3 DRUGS USED IN CORONAVIRUS

 Itolizumab (only preliminary results available)

 Steroids: Dexamethasone, Methylprednisolone

 Low molecular weight Heparin

 Convalescent Plasma Therapy

Official in Not Official in any Pharmacopoeia

Molnupiravir is an orally bioavailable

IJCRT2205468 International Journal of Creative Research Thoughts (IJCRT) www.ijcrt.org e188

Chemical Formula C13H19N3O7

Solubility Soluble in water, Freely Soluble in methanol,

Experimental properties Water solubility 5.77 mg/mL

2.1 INTRODUCTION TO DOSAGE FORM:

Sr. Brand Name Manufacturer Dosage Dose

1 LAGEVRIO Merck Sharp & Capsule Molnupiravir 200 mg

IJCRT2205468 International Journal of Creative Research Thoughts (IJCRT) www.ijcrt.org e189

3.1 METHOD DEVELOPMENT FOR ESTIMATION OF MOLNUPIRAVIR BY

Figure 3.1: UV Spectrum of Molnupiravir (10µg/ml) showing selection of wavelength detection.

B. Preparation of standard solution of Molnupiravir (100 μg/mL):

C. Preparation of standard solution of Molnupiravir (10 μg/mL):

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3.1.3 Preparation of synthetic Mixture and placebo mixture

A. Preparation of synthetic mixture

3.1.4 Selection of Mobile Phase:

A. Preparation of Buffer solution(0.1% Orthophosphoric acid):

D. Preparation of standard solutions (100 μg/mL)

E. Preparation of sample solutions (100 μg/mL)

IJCRT2205468 International Journal of Creative Research Thoughts (IJCRT) www.ijcrt.org e191

Column: Kromasil 100-5-C18 (150 mm x 4.6 mm) 5µ

Mobile Phase: Buffer solution (0.1% Orthophosphoric acid): Acetonitrile

Flow Rate: 1.0 ml/min

Detection Wavelength: 235 nm

Injection volume: 10.0 μl

Run time: 10 min

 Observed values for system suitability test:

Table 3.1 Results for system suitability test.

Theoretical plates per column 8803

Symmetry factor/Tailing factor 1.18

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System suitability / System precision

3.1.2.1 Filter compatibility and saturation

Table 3.2 Filter Compatibility and Saturation

Discarded volume % of Molnupiravir % Difference

3.1.2.2 Linearity & Range

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Preparation of Linearity stock solution (1000 ppm)

Accurately weighed quantity of 50 mg of Molnupiravir standard was transferred to a 50 mL

Y- Intercept was ±2 % of 100 % linearity level response

Figure 3.2: Linearity curve of Molnupiravir

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3.1.2.3 Method precision

Table 3.4 Method precision result

Sample Set % Assay (Molnupiravir)

Preparation of recovery solutions:

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Figure 3.3: Chromatogram of Diluent